In this two phase (R21/R33) project we will develop methods to elucidate cellular signaling mechanisms underlying a defect in central tolerance in the NOD mouse, a model for human Type 1 diabetes. We will compare signal response to TCP stimulation in thymocytes derived from FTOCs on the BDC2.5/NOD and BDC2.5/H-2g7 genetic background. Our investigation will focus on several signal trasduction pathways implucated in negative selection. However, we will also monitor proteome-wide protein phosphorylation events, as it is possible that the mechanism underlying defective clonal deletion will lie in an alternate pathway. In the R21 phase of the project we will develop methodologies which will enable analysis of specific signal transduction pathways and global protein phosphorylation in thymocytes extracted from FTOCs (fetal thymic organ cultures), a model system for the thymus. To identify Akt substrates we will immunoprecipitate with an Akt phosphorylation motifspecific antibody, enzymatically digest immunoprecipitated proteins, and enrich phosphorylated peptides with an Fe3+- charged immobilized metal ion affinity chromatography (IMAC) column prior to LC/MS/MS analysis for identification of specific phosphorylation sites. A similar strategy will be employed for tyrosine phosphorylated proteins, although a panspecific anti-phosphotyrosine antibody will be used for immunoprecipitation. In the R21 phase of the project we will also develop a method enabling identification of protein phosphorylation on a global scale, by extracting proteins from thymocytes, prefractionating the sample at the protein or peptide level, and enriching peptides on the IMAC column prior to LC/MS/MS analysis. In the R33 phase will we apply each of these methods to the analysis of signal transduction in thymocytes extracted from FTOCs following peptide stimulation of the TCR. We will sample the system at specific time points following peptide stimulation and measure relative quantification of protein phosphorylation across the time course. In addition to Akt kinase and phospho-tyrosine mediated signaling, in the R33 phase will investigate phosphorylation state of 14-3-3 binding proteins. Several regulators of negative selection (i.e. HDAC7 and Nur 77) are sequestered to the cytosol following phosphorylation and binding to 14-3-3. By comparing signal transduction pathways responding to peptide stimulation in thymocytes derived from BDC2.5/NOD and BDC2.5/H-2g7 we hope to identify signaling events regulating negative selection of self-reactive thymocytes, with the specific aim of determining the mechanism underlying defective central tolerance in the NOD mouse.